Chlamydia trachomatis is an obligate intracellular pathogen and is the most common cause of bacterial sexually transmitted disease and the leading causative agent of preventable blindness in developing countries. Successful negotiation through the developmental cycle;from the infectious elementary body (EB) to the non-infectious reticulate body (RB) and back to an EB, requires the coordinate expression of defined genes at these specific stages, presumable in response to developmental cues and/or environmental stimuli. The recent identification of a chlamydial specific small RNA (sRNA), IhtA, represents a novel mechanism of gene regulation in Chlamydia. sRNAs are non-coding regulatory elements that coordinate complex biological circuits in response to many different environmental signals by modulating the transcription, stability or translation of a target mRNA. The chlamydial sRNA, IhtA (inhibition of hctA translation), inhibits the translation of Hc1 message, until IhtA is down regulated at the RB to EB differentiation point. Expression of Hc1 causes condensation of the chromosome and is thought to represses global transcription and translation as RBs differentiate to EBs. Thus, IhtA is involved in regulating a critical stage of chlamydial development. We hypothesis that Chlamydia employs sRNAs to rapidly respond to differentiation cues and regulate gene expression during infection. Our overall goal is to understand the role and mechanisms of sRNA regulation of the chlamydial developmental cycle. To this end, the research described in this proposal will explore the mechanism of IhtA function and regulation as well as identify additional sRNAs expressed at differentiation. Aim 1 will determine the regions of both IhtA and Hc1 message critical to IhtA function using a variety of mutants and functional assays. IhtA is rapidly and strongly down regulated at the RB to EB differentiation point, thus regulators of IhtA transcription will also be identified in this aim using biochemical methods. IhtA regulation occurs at a critical decision point of the chlamydial developmental cycle and, as sRNA regulation of gene expression is conserved throughout nature, we hypothesis that additional sRNAs are employed to regulate differentiation. Thus Aim 2 will identify additional sRNAs expressed at the EB to RB and RB to EB transition points by probing custom designed, high density, whole-genome tiling arrays with cDNA synthesized from sRNA fractions. PUBLIC HEALTH RELEVANCE: Chlamydia trachomatis is a bacterial intracellular pathogen of major medical importance as it is the leading cause of preventable blindness in developing countries and the most common bacterially sexually transmitted disease worldwide. Gene regulation in Chlamydia is critical to a successful infection as it controls chlamydial development from the infectious form (EB) to the growth form (RB) and back to the infectious form. Studies undertaken in this proposal will determine mechanisms of gene regulation at both the EB to RB and RB to EB switch, which may provide attractive therapeutic targets.